Molecular Cell Biology Chapter 3. Protein Structure and Function

3.3. Functional Design of Proteins

A key concept in biology is that form and function are inseparable. This concept applies equally well to protein design as to other levels of biological organization (e.g., the morphology of cells and the organization of tissues). In fact, we can often guess how a protein works by looking at its structure. Perhaps the best way to illustrate this is by examining a few protein structures. For instance, a barrel-like nuclear pore, a complex of several proteins, sits in the nuclear membrane and acts as a channel through which molecules travel in or out of the nucleus (Figure 3-20a). In the cavity of a different barrel-like structure, the GroEL/ES chaperonin, protein folding takes place (Figure 3-20b). Some proteins have grooves in their surface, which are logical binding sites for a variety of molecules, especially rod-shaped or filamentous ones. An example is reverse transcriptase, which copies RNA into DNA; this enzyme has a groove on one side through which RNA slides along the surface of the protein (Figure 3-20c). Topoisomerase II, a DNA-binding enzyme, is an articulated enzyme that opens and closes at both ends like locks in a canal (Figure 3-20d). A delight in studying protein structure is uncovering the simple but ingenious ways that nature has built each protein to perform a particular function.

In this section, we examine several features of proteins that are critical to their biological activity and the regulation of that activity, focusing on antibodies, enzymes, and membrane proteins as examples. The functioning of many proteins involves some change in their conformation induced by binding of a specific molecule, change in the environment, or chemical modification. As numerous examples in later chapters will illustrate, such induced conformational changes can make proteins into switches and machines. The changes in conformation can be enormous, as seen in proteins like topoisomerase, an enzyme that moves DNA strands across one another, or myosin, a motor protein that moves along actin filaments.

Proteins Are Designed to Bind a Wide Range of Molecules

The function of nearly all proteins depends on their ability to bind other molecules, or ligands, with a high degree of specificity. As catalysts of chemical reactions, enzymes must first bind tightly and specifically to their target molecules, called substrates, which may be a small molecule (e.g., glucose) or a macromolecule. The many different types of hormone receptors on the surface of cells also display a high degree of sensitivity and discrimination for their ligands, which generally are present at low concentrations in blood. These receptors, essential to signaling between cells, are discussed in Chapter 20.

Two properties of a protein characterize its interaction with ligands. Affinity refers to the strength of binding between a protein and ligand; the equilibrium constant K_eq (Chapter 2) or the dissociation constant K_D for binding is a measure of affinity. Specificity refers to the ability of a protein to bind one molecule in preference to other molecules. Both properties depend on the structure of the ligand-binding site on a protein, which is designed to fit its partner like a mold. For high-affinity and highly specific interactions to occur, the shape and chemical surface of the binding site must be complementary to the ligand molecule. To illustrate this critical concept, we consider how an antibody binds an antigen and how an enzyme catalyzes a chemical reaction.

Antibodies Exhibit Precise Ligand-Binding Specificity

The capacity of proteins to distinguish different molecules is highly developed in blood proteins called antibodies. Animals produce antibodies in response to the invasion of an infectious agent (e.g., a bacterium or a virus) or after exposure to certain foreign substances (e.g., proteins or polysaccharides in pollens). The antibody-inducing agent is called an antigen. The presence of antigen causes an organism to make a large quantity of different antibody proteins, each of which may bind to a slightly different region of the antigen. The constellation of antibodies induced by a given antigen may differ from one member of a species to another.

All antibodies belong to a family of proteins called immunoglobulins. These Y-shaped molecules are formed from two types of polypeptides: heavy chains and light chains. The heavy chains run the length of the molecule; their C-terminal regions pair to form a stem. Visually we can distinguish three globular domains: two identical domains corresponding to each arm and the third composing the stem (Figure 3-21). Each arm of the antibody molecule contains a single light chain linked to a heavy chain by disulfide bonds. The N-terminal regions of both heavy and light chains lie at the tip of each arm and are distinguished by highly variable amino acid sequences. The remaining portions of the sequences in both chains are constant (i.e., nearly identical) among antibodies with different specificities. The arms are the business end of an antibody molecule, since an antigen-binding site lies at the end of each arm. Because of its dimeric structure, each antibody molecule can bind two identical antigen molecules. X-ray crystallographic analysis of antigen-antibody complexes has revealed that the antigenic specificity of an antibody is dependent on three highly variable regions, called complementarity-determining regions (CDRs), near the end of each arm. These regions form the antigen-binding site, which physically matches the antigen like a glove.

Most large antigens have multiple different sites, called epitopes (or antigenic determinants) that can induce production of specific antibodies; each type of antibody binds to its own inducing epitope. For example, lysozyme, an enzyme that degrades the carbohydrate coat of bacteria, induces several different antibodies, each of which binds to a particular epitope on the lysozyme molecule. Although the different epitopes on lysozyme differ greatly in their chemical properties, the interaction between lysozyme and antibody is complementary in all cases; that is, the surface of the antibody's antigen-binding site fits into that of the corresponding epitope as if they were molded together (Figure 3-22). The intimate contact between these two surfaces, stabilized by numerous noncovalent bonds, is responsible for the exquisite binding specificity exhibited by an antibody. Antibodies, for instance, can distinguish between the cells of individual members of a species and in some cases can distinguish between proteins that differ by only a single amino acid. Because of their specificity and the ease with which they can be produced, antibodies are critical reagents in many experiments discussed in the following chapters.

Enzymes Are Highly Efficient and Specific Catalysts

Almost every chemical reaction in a cell is catalyzed by a class of proteins called enzymes. As discussed in Chapter 2, catalysts increase the rates of reactions that are already energetically favorable by lowering the activation energy (see Figure 2-27). In the test tube, catalysts such as charcoal and platinum facilitate reactions but often at high temperatures, at extremes of high or low pH, or in organic solvents. In contrast to these harsh conditions, enzymes must catalyze chemical reactions in the mild conditions of a cell: 37 °C, pH 6.57.5, and aqueous solvents. As we just discussed, all antibodies belong to the immunoglobulin family of proteins and have a similar structure. Enzymes, however, are a structurally diverse group of proteins that have evolved through unrelated and highly divergent mechanisms.

The ability of enzymes to function as catalysts under conditions where nonbiological catalysts would be ineffectual is exemplified by two striking properties: their enormous reaction rates and their specificity. Quite often, the rate of an enzymatically catalyzed reaction is 10⁶10¹² times that of an uncatalyzed reaction under otherwise similar conditions. The specificity of an enzyme denotes its ability to act selectively on one substance or a small number of chemically similar substances, the enzyme's substrates. Like antibody specificity, enzyme specificity depends on a close fit between substrate molecules and their binding sites on an enzyme. An example of specificity is provided by the enzymes that act on amino acids. As noted in Chapter 2, amino acids can exist as two stereoisomers, designated L and D, although only L isomers normally are found in biological systems. Not surprisingly, enzyme-catalyzed reactions involving L-amino acids occur much more rapidly than do those involving D-amino acids, even though both stereoisomers of a given amino acid are the same size and possess the same R groups (see Figure 2-6).

The number of different types of chemical reactions that occur in any one cell is very large: an animal cell, for example, normally contains 10004000 different types of enzymes, each of which catalyzes a single chemical reaction or set of closely related reactions. Certain enzymes are found in the majority of cells because they catalyze synthesis of common cellular products (e.g., proteins, nucleic acids, and phospholipids) or are involved in the production of energy by the conversion of glucose and oxygen to carbon dioxide and water. Other enzymes are present only in a particular type of cell (e.g., a liver cell or a nerve cell) because they catalyze some chemical reaction unique to that cell type. Although most enzymes are located within cells, some are secreted and function in the blood, lumen of the digestive tract, or other extracellular space. Some microbial enzymes are secreted from and are active outside the organism.

An Enzyme's Active Site Binds Substrates and Carries Out Catalysis

Certain amino acid side chains of an enzyme are important in determining its specificity and its ability to accelerate a chemical reaction. In the native conformation of an enzyme, these side chains are brought into proximity, forming the active site. Active sites thus consist of two functionally important regions: one that recognizes and binds the substrate (or substrates), and one that catalyzes the reaction once the substrate has been bound. In some enzymes, the catalytic site is part of the substrate-binding site; in others, the two sites are structurally as well as functionally distinct. The amino acids that make up the active site do not need to be adjacent in the linear polypeptide sequence; rather, folding of the molecule results in juxtaposition of these amino acids, forming a space in which the substrate sits.

To illustrate how the active site binds a specific substrate and then promotes a chemical change in the bound substrate, we examine the action of cAMP-dependent protein kinase (cAPK). This enzyme and other protein kinases, which add a phosphate group to serine, threonine, or tyrosine residues in proteins, are critical for regulating the activity of many cellular proteins. Because the structure of the active site and mechanism of phosphorylation are very similar in all kinases, cAPK can serve as a general model for this important class of enzymes.

As discussed later, an unusual nucleotide called cAMP induces dissociation of the inactive tetrameric form of cAPK, releasing two catalytic subunits. To aid in understanding the mechanism of binding and catalysis, we focus here on the 260-residue "kinase core" of each catalytic subunit. The kinase core, which is largely conserved in all protein kinases, is responsible for the binding of ATP and a target peptide, followed by transfer of a phosphate group from ATP to a serine, threonine, or tyrosine in the peptide. The kinase core consists of a large and small domain with an intervening deep cleft; the active site comprises residues located in both domains.

Substrate Binding by Protein Kinases

The small domain of the kinase core binds ATP, while the large domain binds the target peptide (Figure 3-23). The structure of the ATP-binding site complements the structure of the nucleotide substrate. The adenine ring of ATP sits snugly at the base of the cleft, which is characterized by a highly conserved sequence, Gly-X-Gly-X-X-Gly. This triad of glycine residues, the "glycine lid," is part of a strand-loop-strand motif that closes over the adenine of ATP and holds it in position. The adenine ring sits in a hydrophobic pocket and is positioned by hydrogen bonds and van der Waals attractions with the glycine residues and backbone amide groups. Two invariant residues, lysine at position 72 and aspartic acid at position 184, stabilize the phosphate groups, which protrude from the nucleotide-binding cleft (step 1 in Figure 3-24). Lys-72 bridges to the a and b phosphates of ATP, while the g-phosphate group is chelated by a Mg²⁺ ion bound to Asp-184.

ATP is a common substrate for all protein kinases, but the sequence of the target peptide varies among different kinases. The peptide sequence recognized by cAPK is Arg-Arg-X-Ser-Y, where X is any amino acid and Y is a hydrophobic amino acid. The portion of the polypeptide chain containing the target serine, threonine, or tyrosine is bound to a shallow groove in the large domain of the kinase core. The peptide specificity of cAPK is conferred by several glutamic acid residues in the large domain, which bind the two arginine residues in the target peptide. Different residues determine the specificity of other protein kinases.

Phosphoryl Transfer by cAPK

Figure 3-24 also summarizes the catalytic mechanism of cAPK. Binding of ATP and then the peptide target positions the g phosphate of ATP near the target serine residue of the peptide. Catalysis takes place in two stages. First, a bond forms between the serine and phosphate group, yielding a pentavalent phosphate transition state. Second, the phosphodiester bond between the b and g phosphates is broken, yielding ADP and the phosphorylated peptide. Because the phosphate-serine bond is formed on the opposite side of the phosphodiester bond from the b phosphate, this process is called an in-line mechanism of phosphoryl transfer. The products, ADP and phosphorylated peptide, are then released from the active site.

During catalysis by cAPK, two "catalytic residues" appear to participate in formation of the transition state. Asp-166 is thought to remove a proton from the serine hydroxyl group in the target peptide, while Lys-168 neutralizes the negative charge of the g phosphate. Then the electrons of the deprotonated serine hydroxyl group are thought to form a bond to the g phosphorus atom, yielding the pentavalent transition-state intermediate. The newly created phosphoserine is repelled from the b phosphate of ADP and the catalytic base. The products induce a conformational change in the enzyme, described below, that permits them to diffuse from the active site.

Interactions between residues in the active site of an enzyme and the substrates help stabilize the transition state, thereby allowing more time for the rearrangement of bonds needed to form the products. As explained in Chapter 2, the activation energy is the energy required for formation of the transition state (see Figure 2-27). An enzyme, by virtue of its three-dimensional binding site, reduces the activation energy of a reaction compared with an uncatalyzed reaction involving the same reactants. The ability to bind transition-state intermediates is the one property that distinguishes enzymes from other proteins. If a protein cannot bind a transition-state intermediate, then it cannot catalyze a reaction.

Conformational Changes Induced by Substrate Binding to cAPK

The catalytic subunit of cAPK exists in an "open" and "closed" conformation (Figure 3-25a). In the open position, the large and small domains of the kinase core are separated enough that substrate molecules can bind. Once the active site is occupied by substrate, the domains move together into the closed position. This change in tertiary structure, an example of induced fit, brings the bound target peptide close enough to the terminal phosphate group of the bound ATP that phosphoryl transfer can occur. After the phosphorylation reaction is completed, the presence of the products causes the domains to rotate to the open position, from which the products are released.

The rotation from the open to closed position also causes movement of the short glycine-rich sequence over the ATP-binding cleft in the active site. This small finger of the polypeptide chain, the glycine lid, controls the entry of ATP and release of ADP at the active site. In the open position, ATP can enter and bind to the active site cleft. In the closed position, the glycine-rich sequence moves over the nucleotide and acts as a lid that prevents ATP from leaving (Figure 3-25b). Following phosphoryl transfer, the glycine lid must rotate back to the open position before ADP can be released. Kinetic measurements show that the rate of ADP release is 20-fold slower than that of phosphoryl transfer, reflecting the influence of the glycine lid in cAPK. Mutations in the glycine lid that inhibit its flexibility slow catalysis by cAPK even further. Besides trapping ATP in the binding pocket, the glycine lid prevents water from entering the active site. Water would inhibit the reaction by dampening the charge delocalization steps.

Kinetics of an Enzymatic Reaction Are Described by V_max and K_m

Enzymatic specificity is usually quantified in relative terms; that is, the reaction with a good substrate may occur, for example, 10,000 times faster than it does with a poor substrate. The catalytic action of an enzyme on a given substrate can be described by two parameters: K_m (the Michaelis constant), which measures the affinity of an enzyme for its substrate, and V_max, which measures the maximal velocity of the reaction at saturating substrate concentrations. Equations for K_m and V_max are most easily derived by considering the simple reaction

in which the rate of product formation v depends on the concentration of substrate, [S], and on the concentration of the enzyme, [E].

For an enzyme with a single catalytic site, Figure 3-26a shows how the rate of product formation depends on [S] when [E] is kept constant. At low concentrations of S, the reaction rate is proportional to [S]. As [S] is increased, the rate does not increase indefinitely in proportion to [S]; rather, it eventually reaches a maximum velocity V_max. The value of V_max is independent of [S], but is proportional to [E] and to the catalytic constant k_cat, which is an intrinsic property of the individual enzyme. Halving [E] reduces the reaction rate at all values of [S] by half. Both V_max and K_m for a particular enzyme and substrate can be determined from experimental curves of reaction velocity versus substrate concentration, as illustrated in Figure 3-26.

When interpreting kinetic curves such as those in Figure 3-26, bear in mind that all enzyme-catalyzed reactions include at least three steps: (1) the binding of a substrate (S) to an enzyme (E) to form an enzyme-substrate complex (ES), (2) the conversion of ES to the enzyme-product complex (EP), and (3) the release of the product (P) from EP, to yield free P:

In the simplest case, when the release of P is very rapid, we can simplify the reaction equation as follows:

In this case, the rate of product formation v is equal to k_cat × [ES]. Starting from this relationship, we can derive the Michaelis-Menten equation

where K_m, the Michaelis constant, is defined as (k₂+k_cat)/k₁. This equation fits the curves shown in Figure 3-26.

The slowest step in most enzymatic reactions is conversion of the enzyme-substrate complex ES to the free enzyme E and product P. In such cases, k_cat is much less than k₂, so that

where K_d is the dissociation constant for binding of S to E. Thus the parameter K_m describes the affinity of an enzyme for its substrate. The smaller the value of K_m, the more avidly the enzyme can bind the substrate from a dilute solution and the smaller the concentration of substrate needed to reach half-maximal velocity (see Figure 3-26b). The concentrations of the various small molecules in a cell vary widely, as do the K_m values for the different enzymes that act on them. Generally, the intracellular concentration of a substrate is approximately the same as or greater than the K_m value of the enzyme to which it binds.

Many Proteins Contain Tightly Bound Prosthetic Groups

The native conformation and activities of some proteins require the presence of a prosthetic group, a small nonpeptide molecule or metal that binds tightly to a protein, keeping the protein in a fixed conformation and participating in binding ligands. For example, each of the four subunits of hemoglobin binds and enfolds a prosthetic group called heme, which consists of an iron atom held in a cage by protoporphyrin:

The heme groups are the oxygen-binding components of hemoglobin (see Figure 3-11). Heme is also present in the cytochromes of the electron-transport chain; in this case, it functions to bind electrons. Other electron-transport proteins employ sulfur or flavin as prosthetic groups. In addition to acting as carriers of oxygen or electrons, prosthetic groups can act as antennae. For example, proteins involved in vision or photosynthesis contain retinal or chlorophyll, which absorb energy from sunlight. Prosthetic groups can be linked to proteins noncovalently, as in hemoglobin, or covalently, as in cytochrome.

The activity of numerous enzymes also depends on the presence a prosthetic group, commonly referred to as a coenzyme. Many coenzymes act to lower the activation energy of biochemical reactions by forming a covalent intermediate with a substrate. For instance, the enzyme that converts the amino acid histidine into histamine (a potent dilator of small blood vessels) requires the coenzyme pyridoxal phosphate. In this reaction, a covalent bond first forms between histidine and the enzyme-bound pyridoxal phosphate, forming a Schiff base intermediate. Rearrangement of the bonds in this intermediate yields carbon dioxide, which is released, and a second intermediate. This is then hydrolyzed, producing the product, histamine, and regenerating the coenzyme, pyridoxal phosphate.

A Variety of Regulatory Mechanisms Control Protein Function

Most reactions in cells do not occur independently of one another or at a constant rate. Instead, the catalytic activity of enzymes is so regulated that the amount of reaction product is just sufficient to meet the needs of the cell. As a result, the steady-state concentrations of substrates and products will vary depending on cellular conditions. The flow of material in an enzymatic pathway is controlled by several mechanisms, some of which also regulate the functions of nonenzymatic proteins.

One of the most important mechanisms for regulating protein function entails allosteric transitions, changes in the tertiary and/or quaternary structure of a protein induced by binding of a small molecule, which may be an activator, inhibitor, or substrate. Allosteric regulation is particularly prevalent in multimeric (multisubunit) enzymes. Some multimeric enzymes are composed of identical subunits, each containing an active site and, often, a distinct regulatory site. Other enzymes comprise structurally different subunits; in these, active sites and regulatory sites may be located on different subunits.

Allosteric Release of Catalytic Subunits

As mentioned previously, cAMP-dependent protein kinase (cAPK) exists as an inactive tetrameric protein composed of two catalytic subunits and two regulatory subunits. Each regulatory subunit contains a pseudosubstrate sequence that binds to the active site in a catalytic subunit. By blocking substrate binding, the regulatory subunit inhibits the activity of the catalytic subunit. Binding of the allosteric effector molecule cyclic AMP (cAMP) to the regulatory subunit induces a conformational change in the pseudosubstrate sequence so that it no longer can bind the catalytic subunit. Thus the inactive tetramer dissociates into two monomeric active catalytic subunits and a dimeric regulatory subunit (Figure 3-27). As discussed in Chapter 20, binding of various hormones to cell-surface receptors induces a rise in the intracellular concentration of cAMP, leading to activation of cAPK. Once the signaling ceases and the cAMP level decreases, the activity of cAPK is turned off by reassembly of the inactive tetramer.

Allosteric Transition between Active and Inactive States

Many multimeric enzymes undergo allosteric transitions that alter the relationship of the subunits to one another but do not cause dissociation as in cAPK. A wellunderstood enzyme illustrating this mechanism is aspartate transcarbamoylase (ATCase). This bacterial enzyme catalyzes the first step in the pyrimidine biosynthetic pathway:

ATCase, which is composed of six catalytic subunits and six regulatory subunits, exists in an active R state and inactive T state (Figure 3-28). The equilibrium between these states is shifted toward the inactive T state by binding of cytidine triphosphate (CTP), an end product of the pyrimidine pathway, to the regulatory subunits. Thus CTP is an allosteric inhibitor of ATCase. The CTP-induced allosteric transition in ATCase is an example of feedback inhibition, whereby an enzyme that catalyzes an early reaction in a multistep pathway is inhibited by an ultimate product of the pathway. Clearly, this type of regulation prevents accumulation of pyrimidines in excess of what the cell needs for DNA synthesis.

The mechanism of feedback inhibition helps regulate most biosynthetic pathways; that is, the final product of the pathway inhibits the enzyme that catalyzes the first step, thus preventing both production of the intermediate products and unnecessary metabolic activity. Feedback inhibition of enzyme function is reversible. If the concentration of free feedback inhibitor (e.g., CTP) falls, the bound inhibitor dissociates from the regulated enzyme, which then reverts to its active conformation. The binding of a feedback inhibitor to an enzyme and its subsequent release can be described by the equilibrium binding constant K_i, which is similar to the Michaelis constant K_m used to describe substrate binding.

Cooperative Binding of Ligands

In many cases, especially when a protein binds several molecules of one ligand, the binding is graded; that is, binding of one ligand molecule affects the binding of subsequent ligand molecules. Such cooperative allostery, or cooperative binding, permits many multisubunit proteins to respond more efficiently to small changes in ligand concentration than would otherwise be possible. In positive cooperativity, sequential binding is enhanced; in negative cooperativity, sequential binding is inhibited.

Hemoglobin presents a classic example of positive cooperative binding. Each of the four subunits in hemoglobin can bind one oxygen molecule. Binding of oxygen to one subunit induces a local conformational change whose effect spreads to the other subunits, lowering the K_m for binding of additional oxygen molecules.

Many multimeric enzymes, including aspartate transcarbamoylase (ATCase), also exhibit cooperative binding of substrate. For reactions catalyzed by such enzymes, a plot of reaction velocity versus substrate concentration yields a sigmoidal curve rather than the hyperbolic curve characteristic of enzymes with typical Michaelis-Menten kinetics. As a result of cooperative substrate binding, the maximal enzyme activity (V_max) is achieved over a narrow range of substrate concentration (Figure 3-29). Other multimeric enzymes exhibit cooperative binding of an allosteric inhibitor. Because of cooperative allostery, a quite small change in ligand concentration can effectively turn an enzyme on or off.

Cyclic Protein Phosphorylation and Dephosphorylation

As noted earlier, one of the most common mechanisms for regulating protein activity is the addition and removal of phosphate groups from serine, threonine, or tyrosine residues. Protein kinases catalyze phosphorylation, and phosphatases catalyze dephosphorylation. Although both reactions are essentially irreversible, the counteracting activities of kinases and phosphatases provide cells with a "switch" that can turn on or turn off the function of various proteins (Figure 3-30). Phosphorylation changes a protein's charge and generally leads to a conformational change; these effects can significantly alter ligand binding by a protein and its activity.

Nearly 3 percent of all yeast proteins are protein kinases or phosphatases, reflecting the importance of phosphorylation and dephosphorylation reactions even in simple cells. These enzymes target all classes of proteins including structural proteins, enzymes, membrane channels, and signaling molecules. In later chapters, we will encounter many examples of important cellular functions that are controlled by phosphorylation and dephosphorylation of specific proteins.

Proteolytic Activation

The regulatory mechanisms discussed so far can act like switches, turning proteins on and off. Regulation of some enzymes involves the irreversible activation of an inactive form, commonly at the site where the active enzyme is needed. Good examples of such enzymes are the digestive proteases trypsin and chymotrypsin, which are synthesized in the pancreas and secreted into the small intestine as inactive precursors, or zymogens, called trypsinogen and chymotrypsinogen, respectively. In the protease-rich environment of the small intestine, the zymogens are converted to the active enzymes, which then begin to hydrolyze the peptide bonds of ingested proteins. As shown in Figure 3-31, two irreversible proteolytic cleavages of chymotrypsinogen yield active chymotrypsin. The delay in activation of these proteases until they reach the intestine prevents them from digesting the pancreatic tissue in which they are made.

Other Regulatory Mechanisms

The activities of enzymes are extensively regulated in order that the numerous enzymes in a cell can work together harmoniously. All metabolic pathways are closely controlled at all times. Synthetic reactions occur when the products of these reactions are needed; degradative reactions occur when molecules must be broken down. All the regulatory mechanisms described above affect enzymes locally at their site of action.

Regulation of cellular processes, however, involves more than simply turning enzymes on and off. Some regulation is accomplished by keeping enzymes in compartments where the delivery of substrate or exit of product is controlled. In many cases, the compartments are organelles, such as the mitochondria, nuclei, or lysosomes. Compartmentation permits competing reactions to occur simultaneously in different parts of a cell. In addition to compartmentation, cellular processes are regulated by enzyme synthesis and destruction. Often enzymes are synthesized at low rates when the cell has no need for their activities; however, upon increased demands by the cell (for instance, appearance of substrate), new enzyme is synthesized. Later, the pool of enzyme is lowered when levels of substrate decrease or the cell becomes inactive.

SUMMARY


The function of nearly all proteins depends on their ability to bind other molecules (ligands). Ligand-binding sites on proteins and the corresponding ligands are chemically and topologically complementary. The affinity of a protein for a particular ligand refers to the strength of binding; its specificity, to the restriction of binding to one or a few preferred ligands.

Enzymes are catalytic proteins that accelerate the rate of cellular reactions by lowering the activation energy and stabilizing transition-state intermediates.

Enzyme active sites comprise two functional parts: a substrate-binding region and a catalytic region. The amino acids composing the active site are not necessarily adjacent in the amino acid sequence, but are brought into proximity in the native conformation.

The kinetics of many enzymes are described by the Michaelis-Menten equation. From plots of reaction rate versus substrate concentration, two characteristic parameters of an enzyme can be determined: the Michaelis constant K_m, a measure of the enzyme's affinity for substrate, and the maximal velocity V_max (see Figure 3-26).

Many multimeric enzymes and other proteins exhibit allostery. In this phenomenon, binding of one ligand molecule (a substrate, activator, or inhibitor) induces a conformational change, or allosteric transition, that alters the protein's activity or affinity for other ligands.

In multimeric proteins that bind multiple ligands, binding of one ligand molecule may increase or decrease the binding affinity for subsequent ligand molecules. Enzymes that cooperatively bind substrates exhibit sigmoidal kinetics (see Figure 3-29).

Allosteric mechanisms can act like switches, turning protein activity on and off. Cyclic phosphorylation and dephosphorylation of amino acid side chains can have the same regulatory effect. Proteolytic cleavage irreversibly converts inactive zymogens into active enzymes.